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研究生:洪毓均
研究生(外文):Yu-Chun Hung
論文名稱:烯雙炔抗腫瘤抗生素生色團蛋白水解蛋白活性之探討
論文名稱(外文):Proteolytic activity study on enediyne antitumor antibiotic chromoproteins
指導教授:金德航
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學系所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:105
中文關鍵詞:烯雙炔抗腫瘤抗生素水解蛋白活性可導素啶新抑癌素
外文關鍵詞:enediyne antitumor antibioticproteolytic activityKedarcidinneocarzinostatin
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天然產的含有烯雙炔的抗腫瘤抗生素具破壞核酸之能力,能有效殺死腫瘤細胞。其中可導素啶和新抑癌素均屬於生色團蛋白複合體,由不穩定的小分子藥物-烯雙炔生色團,及保護生色團的攜帶蛋白所組成。二者以非共價鍵的形式緊密結合。
有研究報導可導素啶和新抑癌素的攜帶蛋白,也兼具蛋白酶的功能,可水解染色質上的組蛋白;但後來又有研究報導新抑癌素水解組蛋白的活性是來自於其它的污染物,與新抑癌素攜帶蛋白無關。
新抑癌素是目前探討最廣泛的一種烯雙炔抗腫瘤抗生素,相較之下,可導素啶仍有許多性質和機制尚未被研究,目前尚未確證可導素啶蛋白是否真正具有蛋白酶的功能;本論文探討這二種抗生素在水解蛋白活性上之差異,並嘗試研究該活性的來源。
我們培養放線菌並純化了天然可導素啶生色團蛋白複合體,也分離並純化了其攜帶蛋白與活性生色團。為了比較對照水解組蛋白的功能,我們也培養大腸菌並純化了重組可導素啶攜帶蛋白,同時純化了天然和重組新抑癌素蛋白。利用蛋白質電泳、紫外光-可見光光譜儀、高效能液向層析、圓二色旋光光譜儀及質譜儀等,一一分析鑑定純化完成的蛋白,最後利用化學法來鑑定雙硫鍵的存在,確認了純化出正確的可導素啶及新抑癌素蛋白樣品。
純化的蛋白進行水解組蛋白的實驗後,發現可導素啶蛋白和新抑癌素蛋白,不論天然的或重組的,均不具有蛋白酶活性,此活性物質可藉由純化的方式順利移除。相較於純化的蛋白,天然可導素啶和新抑癌素生色團蛋白複合體的溶液皆具有水解組蛋白的能力,推測溶液中存在著具有蛋白酶功能的活性物質,而可導素啶溶液中的活性物質,水解組蛋白的效率較高。
為了探討水解蛋白活性是否與生色團有關聯,我們取重組新抑癌素蛋白加上萃取的生色團,形成再結合的新抑癌素生色團蛋白複合體,之後進行組蛋白水解反應,我們發現該重組的新抑癌素生色團蛋白複合體不具有水解蛋白的能力;但是再結合的可導素啶可發現些微的活性。我們目前尚無法確知該活性的來源,僅知該活性物質水解蛋白的功能,在低溫環境下作用能力較差,經加熱處理後會喪失活性,也會因環境中的酸鹼值改變而影響水解組蛋白的能力;但不受到硫醇和無氧環境的影響。
Kedarcidin (KDC) and neocarzinostatin (NCS) are potent antitumor antibiotic chromoproteins, composed of an enediyne-containing chromophore embedded in a highly acidic single chain polypeptide apoprotein. These chromoproteins have been reported to possess protease activity against histone proteins and a wide-range of other target proteins. As enediyne antibiotic chromoproteins are well known to cause chromosomal damages through radical based DNA-cleavage activity related to the bound chromophores, the additional histone proteolytic activity holds critical importance on its mechanism of action.
Secreted natural KDC chromoprotein was purified from cultures of actinomycete strain L585-6 (ATCC 53650). We found histone-specific proteolytic activity from the preparation of natural KDC and NCS (gift from Kayaku Ltd., Tokyo, Japan) were similar to the activity described in the previous reports, in which the KDC hydrolyses histone H1 more efficiently than the NCS, as observed in time-course experiment. With the aim of dissecting the molecular basis of proteolytic activity of KDC and NCS, Escherichia coli expression systems allowing efficient production of apo-KDC and apo-NCS were previously constructed. The purified recombinant proteins were properly folded and functional. In an attempt to distinguish whether the H1-proteolytic activity comes from either apoprotein, chromophore or an external species present in the chromoprotein preparations, we performed series of experiments using individual components of chromoproteins in the proteolytic assay. We first unambiguously demonstrate that both natural and recombinant apo-KDC and apo-NCS have no detectable protease activity, while incubated individually with the histone-H1. Methanol extracted NCS chromophore and HPLC purified KDC chromophore also lack proteolytic function under similar assay conditions. Contrary to the natural chromoprotein preparation, the reconstituted NCS consisting of 1:1 molar ratio of recombinant apoNCS and NCS chromophore shows no detectable proteolytic activity, whereas the reconstituted KDC reveals minor proteolytic activity. The results indicate that the proteolytic activity is unlikely related to the chromoprotein components, and the minor proteolytic activity in the reconstituted KDC might be due to retention of some contaminating species. We also found that high temperature, acidic pH or anaerobic environment prevents the proteolytic activity. However, addition of thiols shows no detectable influence on the same.
This investigation improves our understanding on the seemingly elusive proteolytic activity of the enediyne chromoprotein antibiotics. These results suggest that the possible proteolytic activity of the proteins of this family should be critically reconsidered.
中文摘要 i
英文摘要 iii
目次 v
圖表目錄 vii
縮寫表 xi
第一章 緒論 1
(一) 烯雙炔抗腫瘤抗生素 1
(二) 新抑癌素的發現 4
(三) 新抑癌素蛋白的結構與保護機制 6
(四) 新抑癌素生色團的結構與反應機制 9
(五) 可導素啶之簡介 13
(六) 烯雙炔抗腫瘤抗生素的水解蛋白活性與研究動機 18
第二章 材料與方法 22
(一) 天然新抑癌素和新抑癌素生色團的來源 22
(二) 重組質體DNA之轉殖作用 22
(三) 純化質體DNA 25
(四) 質體DNA之電泳分析 25
(五) DNA定序 26
(六) 重組蛋白的表現及純化 26
(七) 天然可導素啶之純化 29
(八) 天然新抑癌素蛋白之純化 32
(九) 蛋白質電泳分析 32
(十) 高效能液相層析純化可導素啶生色團與蛋白 32
(十一) 蛋白質雙硫鍵完整性之鑑定 33
(十二) 圓二色光譜分析 33
(十三) 生色團與重組蛋白之再結合反應 34
(十四) 水解蛋白活性之反應 34
第三章 結果與討論 36
(一) 重組可導素啶與重組新抑癌素蛋白基因之製備 36
(二) 重組可導素啶蛋白與重組新抑癌素蛋白的取得與純化 36
(三) 天然可導素啶的取得與純化 38
(四) 天然可導素啶蛋白和新抑癌素蛋白的取得與純化 39
(五) 天然可導素啶之純度鑑定與可導素啶生色團的取得 40
(六) 圓二色光譜分析 40
(七) 鑑定蛋白質分子量及雙硫鍵之完整性 41
(八) 天然可導素啶和新抑癌素之生物活性,以及其攜帶蛋白殘餘活 性之測試 42
(九) 可導素啶和新抑癌素水解Histone H1之反應 43
第四章 結論與未來展望 47
實驗結果圖表 50
參考文獻 97
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